High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed.     

Evidence for linkage position determination in known feruloylated mono- and disaccharides using electrospray ion trap mass spectrometry

Various feruloylated arabinose- and galactose-containing mono- and disaccharides with known linkage configurations (2-O-(trans-feruloyl)-L-arabinopyranose, 5-O-(trans-feruloyl)-L-arabinofuranose, O-[2-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->5)-L-arabinofuranose, and O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose) were analyzed by electrospray ionization mass spectrometry using an ion trap or a quadrupole time-of-flight (Q-TOF) mass analyzer. Collision-induced dissociation (CID) experiments using the two mass analyzers generated similar tandem mass spectrometric (MS/MS) fragmentation patterns. However, the ester-bond cleavage ions were more abundant using the Q-TOF mass analyzer. Compared with the positive ion mode, the negative ion mode produces simpler and more useful CID product-ion patterns. For arabinose-containing feruloylated compounds, results obtained with both analyzers show that it is possible to assign the location of the feruloyl group to the O-2 or O-5 of arabinosyl residues. In the characterization of the 2-O-feruloyl and 5-O-feruloyl linkages, the relative abundance of the cross-ring fragment ions at m/z 265 (-60 u or -62 u after 18O-labelling) and at m/z 217 (-108 u or -110 u after 18O-labelling) play a relevant role. For galactose-containing feruloylated compounds, losses of 60, 90 and 120 Da observed in MS3 experiment correspond to the production of 0,2A1, 0,3A1 and (0,2A1-60 Da) cross-ring cleavage ions, respectively, fixing the location of feruloyl group at the O-6 of the galactose residue.     

Multiplex multidimensional nanoLC-MS system for targeted proteomic analyses

The present work describes a dual-column and dual-sprayer LC-MS system for high-throughput proteomic analyses. This system consists of two precolumns for sample desalting and two analytical columns. Each column is terminated by a nanoelectrospray emitter mounted on a robotic arm enabling their sequential positioning in front of the sampling cone of the mass spectrometer. The effluent from each emitter is recorded in separate acquisition channels without detectable crosstalk. Gradient elution to both nanoLC columns is delivered by a single HPLC system via a flow splitter. The reproducibility of retention time and peak intensity of the present multiplex system were comparable to those obtainable using a single emitter configuration. Replicate injections of complex tryptic digests (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity on both columns with RSD values of less than 0.9 and 18.6%, respectively. The application of this system is demonstrated for the monitoring of protein expression changes in U937 human monocyte cells with and without phorbol ester administration. Furthermore, we also demonstrated the use of this multiplex system in a 2-D LC configuration to increase sample loading and throughput for the analysis of biomarker samples of higher complexity. Variations in peptide abundance down to two-fold change were identified across salt fractions for spiked tryptic digests present at a level of 50 fmol in 1.5 microg of plasma samples.     

Charge state dependent collision-induced dissociation of native and reduced porcine elastase

The [M + 20H](20+)-[M + 12H](12+) charge states of native and reduced porcine elastase, a 25.9 kDa serine protease, were subjected to collisional activation in a quadrupole ion trap. For most charge states, ion parking was used to increase the number of parent ions over that yielded directly by electrospray. Ion-ion proton transfer reactions were used to reduce product ion charge states largely to +1 to simplify spectral interpretation. Both forms of the protein show charge state dependent fragmentation behavior. The native protein, which contains four disulfide linkages, shows almost no evidence for fragmentation within the regions of the protein linked by disulfide bonds. However, at the lowest charge states studied, evidence for cleavage of a least one of the disulfide bonds was evident in the appearance of a c-type ion. The highest charge states of native elastase showed several prominent cleavages C-terminal to valine residues. As the charge state decreased, however, preferential cleavages at acidic amino acid residues became important. The reduced form of the protein did not show particularly prominent cleavages at valine residues. However, many of the same preferential cleavages at acidic amino acid residues noted for the native protein were also observed in the same charge states of the reduced protein. The reduced protein also showed additional cleavages from regions of the protein that are ordinarily protected by disulfide linkages in the native form.     

基于遗传算法的多通道密集式移动货架订单拣选研究

密集式移动货架越来越多地应用到仓储实践中,提高了仓储空间利用率,但增加了订单拣选的时间成本。本文根据密集式移动货架的仓储布局特点,针对多条通道可同时打开的情况,将货架移动时间转换成通道移动距离进行计算,提出了多条通道依次移动的优化规则,以整批订单拣选所耗费的总时间最少为目标,建立了订单拣选顺序优化的数学模型。针对该模型的特点,设计了实数编码且全局寻优的遗传算法,并进行了不同规模的算例模拟。计算结果表明,该算法具有较强的适用性,针对不同规模的问题,均有显著的优化效果;货架数量、订单数量以及移动通道数量的小幅度增减,将会导致总拣选时间较大幅度的波动;多条移动通道初始位置居于中部或均匀分散,总拣选时间略优于其集中于仓储系统一端。     

The Structure of an Archaeal B‐Family DNA Polymerase in Complex with a Chemically Modified Nucleotide

Archaeal B‐family DNA polymerases (DNA pols) are the driving force of cutting‐edge biotechnological applications like next‐generation sequencing. The acceptance of chemically modified nucleotides by DNA pols is key to these technologies. Until now, no structural data have been available for these DNA pols in complex with modified substrates, which could build the basis for understanding interactions between the enzyme and the chemically modified nucleotide and for the further development of next‐generation nucleotides. For the first time, we crystallized an exonuclease‐deficient variant of the wild‐type B‐family KOD DNA pol with a modified nucleotide in a closed, ternary complex. We also crystalized the A‐family DNA pol KlenTaq with the same nucleotide. The reported structural data reveal how the protein and the DNA modulate two distinct conformations of the appended moiety in the A‐ and B‐family DNA pols and how these influence the processing of the modified nucleotide. Overall, this study provides first insight into the interplay between B‐family DNA pols and relevant modified substrates.     

Mixed dye degradation by Bacillus pseudomycoides and Acinetobacter haemolyticus isolated from industrial effluents: A combined affirmation with wetlab and in silico studies

The environment-friendly bacterial strains are used for wastewater treatment due to their high degrading capability and cost-effectiveness. In the present study, we isolated, identified, characterized, and optimized culture condition ( Temperature 35 °C for both, time up to 96 h, pH 7 and 7.5 respectively) of two dye degrading bacteria from industrial effluents. Morphological, biochemical, and molecular identification confirmed those strains as Bacillus pseudomycoides and Acinetobacter haemolyticus. Spectrophotometric methods were used to investigate the dye degradation(single and mixed dye) capability of these two bacteria. These strains were the potential for degrading methylene green (MG), basic violet (BV) and acid blue (AB) dyes. In case of MG + BV, B. pseudomycoides, and A. haemolyticus showed a degradation rate of 74% and 75%, respectively. While degradation was found 75% and 82% for MG + AB combination, 73% and 73% for AB + BV combination, and 80% and 82% for MG + BV + AB combination respectively. Azoreductase enzymes from bacteria are essential for breaking down the azo bond in textile azo dyes. In molecular docking, the binding energy of three docking complexes (protein and MG, protein and BV, protein and AB) were ?6.3, ?6.6, and ?6.8 Kcal/mol, respectively. The binding stability of the docked complexes was ensured by the root mean square deviations (RMSD), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bond in a molecular dynamics simulation study, indicating strong and stable binding. This study revealed that both B. pseudomycoides and A. haemolyticus could decolorize single and mixed dyes efficiently. As a result, both the strains could be used in further research to apply their potentiality in large-scale dye degradation in the future.     

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation (“CID”) and beam-type CID also termed as higher-energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.